Molecular and Biochemical Evidence of the Toxic Effects of Terbuthylazine and Malathion in Zebrafish

Abstract

Our research sought to determine the molecular and biochemical effects of environmentally relevant exposure to commonly used chloro-s-triazine herbicide terbuthylazine and organophosphate insecticide malathion on zebrafish. To this aim, mature zebrafish were exposed to 2 and 30 µg L^-1 terbuthylazine and 5 and 50 µg L^-1 malathion alone and in combination for 14 days. Aside from the accumulation of TBARS and protein carbonyls, a decrease in antioxidants and succinate dehydrogenase activity, an increase in oxidized glutathione, and enhanced apoptosis via Caspase-3 and BAX overexpression were observed. Furthermore, terbuthylazine and malathion induced mitochondrial swelling (up to 210% after single exposure and up to 470% after co-exposure) and lactate dehydrogenase leakage (up to 268% after single exposure and up to 570% after co-exposure) in a concentration-dependent manner. Significant upregulation of ubiquitin expression and increased cathepsin D activity were characteristics that appeared only upon terbuthylazine exposure, whereas the induction of IgM was identified as the specific characteristic of malathion toxicity. Meanwhile, no alterations in the zebrafish hypothalamic-pituitary-thyroid axis was observed. Co-exposure increased the adverse effects of individual pesticides on zebrafish. This study should improve the understanding of the mechanisms of pesticide toxicity that lead to fish impairment and biodiversity decline.

Keywords: Danio rerio; apoptosis; chloro-s-triazine herbicide; cytotoxicity; immune toxicity; organophosphate insecticide; oxidative stress.

Figure 1

Effects of terbuthylazine (2 µg L⁻¹ (TL) and 30 µg L⁻¹ (TH)) and malathion (5 µg L⁻¹ (ML) and 50 µg L⁻¹ (MH)) exposures on antioxidants status in the liver of zebrafish. (A) Total antioxidant capacity; (B) catalase; (C) Glutathione total; (D) Glutathione oxidized. When columns share the different letters (a–f), they represent significantly different values (p < 0.05). The means and the standard errors of the mean are presented. N = 6.

Figure 2

Effects of terbuthylazine (2 µg L⁻¹ (TL) and 30 µg L⁻¹ (TH)) and malathion (5 µg L⁻¹ (ML) and 50 µg L⁻¹ (MH)) exposures on reactive oxygen and nitrogen species as well as oxidative lesions in the liver of zebrafish. (A) Reactive oxygen species; (B) Nitric oxide; (C) TBA-reactive substances; D, Protein carbonyls. When columns share the different letters (a–e), they represent significantly different values (p < 0.05). The means and the standard errors of the mean are presented. N = 6.

Figure 3

Effects of terbuthylazine (2 µg L⁻¹ (TL) and 30 µg L⁻¹ (TH)) and malathion (5 µg L⁻¹ (ML) and 50 µg L¹ (MH)) exposures on immune (A) and endocrine (B,C) traits in zebrafish blood and mitochondrial (D,E) and lysosome (F) related indices in hepatocytes. Columns with different letters have significantly different values (p < 0.05). The means and the standard errors of the mean are presented. N = 6.

Figure 4

Effects of terbuthylazine (2 µg L⁻¹ (TL) and 30 µg L⁻¹ (TH)) and malathion (5 µg L⁻¹ (ML) and 50 µg L⁻¹ (MH)) exposures on the ratio of DNA strand breaks in hepatocytes and lactate dehydrogenase leakage in blood of zebrafish. (A) DNA strand breaks; (B) Lactate dehydrogenase. Columns with different letters (a–e) have significantly different values (p < 0.05). The means and the standard errors of the mean are presented. N = 6.

Figure 5

Effects of terbuthylazine (2 µg L⁻¹ (TL) and 30 µg L⁻¹ (TH)) and malathion (5 µg L⁻¹ (ML) and 50 µg L⁻¹ (MH)) exposures on mRNA expression of apoptotic, oxidative stress, and DNA damage and repair markers in the liver of zebrafish. (A) the nuclear factor erythroid 2–related factor 2, (B) Ubiquitin; (C) Caspase 3; (D) DNA repair protein RAD51; (E) B-cell lymphoma protein 2 (Bcl-2)-associated X; (F) B-cell lymphoma protein 2. Columns with different letters have significantly different values (p < 0.05). The means and the standard errors of the mean are presented. N = 6.

Figure 6

Integrative oxidative stress response and biomarker response in zebrafish after the effects of terbuthylazine (2 µg L⁻¹ (TL) and 30 µg L⁻¹ (TH)) and malathion (5 µg L⁻¹ (ML) and 50 µg L⁻¹ (MH)). IBR- integrative index of biomarker response, IOS – integrative index of oxidative stress.